The long-term goal of this project is to investigate a novel function of the Hu family of paraneoplastic neurological disease (PND) antigens in neurons as alternative splicing regulators. Hu proteins belong to a group of neuronal RNA-binding proteins that were cloned using antiserum from PND patients and are antigens of the Hu syndrome triggered by small cell lung carcinomas. Similar to their closest Drosophila homolog ELAV, Hu proteins were shown to be required for neuronal differentiation in the mammalian nervous system. However, the function of Hu proteins as RNA-binding proteins is not understood. Except for a handful of examples indicating that they bind to the AU-rich element of 3'-untanslated region of mRNA to regulate mRNA stability and translation, the in vivo targets of Hu proteins are largely unknown. Recently we have identified two RNA targets for Hu proteins and provided compelling evidence for a prominent role of these proteins as alternative splicing regulators. To characterize the novel function of Hu proteins as splicing regulators in neurons, we propose three specific aims. In aims I and II, we will study the mechanisms that control the neuron-specific alternative RNA processing of the two newly identified Hu target-containing pre-mRNAs by Hu proteins. Specific hypotheses regarding the function of Hu proteins will be generated and tested with both in vitro and in vivo experiments. In aim III, we will identify additional neuronal targets bound by Hu proteins using both in silico database screening and genomic SELEX approaches. The identification of these novel targets will provide the basis for future work that will focus on universality of the principle developed in the proposed studies. These studies will provide not only significant insights into the role of Hu proteins in the development and function of neurons, but also important hints of pathogenesis of the PND disease, the Hu syndrome. Finally, these studies will also greatly increase our understanding of the mechanisms controlling alternative splicing in neurons.